Both the brain and the pituitary gland have been known to contain mitogenic factors for cultured cells; however, it was unclear what the relationship was with classical pituitary hormones, such as TSH, LH, FSH, GH and ACTH, until the identification in the pituitary gland in 1974 of a growth factor called fibroblast growth factor (FGF). The presence of two forms of FGF, acidic and basic, has now been widely acknowledged by many specialists and research groups working in the fibroblast growth factor area. A thorough description of the events leading to the discovery of the acidic and basic forms of FGF during the period of 1973 to 1978 is contained in Molecular Cellular Biochemistry, 25, 79-110 (1979).
The acidic form of FGF was first discovered and identified in 1975, Gospodarowicz, D., Nature, 256, 216-219 (1975). At the time of this discovery, Dr. Gospodarowicz was working with myoblasts and accordindly the term myoblast growth factor was used. Purification of an acidic brain GF is also described in U.S. Pat. No. 4,444,760(Apr. 24, 1984).
In 1974, the identification in the pituitary gland of a growth factor called fibroblast growth factor
(FGF) was reported which was shown to be distinct from pituitary hormones, Gospodarowicz, D., Nature, 249, 123-127 (1974). This growth factor was demonstrated to be a basic protein (pI of 9.6),to be a potent mitogen for either normal diploid fibroblasts or established cell lines, and to have a molecular weight of about 14,000. Subsequent to this discovery, the basic nature of brain and pituitary derived FGFs has been described in further detail in numerous publications, Gospodarowicz et al., J. of Biol. Chem., 253, 3736-3743 (1978); Gospodarowicz et al., J. of Biol. Chem, 253, 1226-1276 (1982); Gospodarowicz et al., J. of Cell. Physiol., 122, 323-332 (1985); Lemmon et al., J. of Cell. Biol., 95, 162-169; (1982) Gliwin & Haushcka, Biochemistry, 25, 3487-3492 (1986).
Later studies have confirmed that, in addition to fibroblasts, basic FGF is also mitogenic for a wide variety of normal diploid mesoderm-derived and neurocrest-derived cells including granulosa cells, adrenal cortical cells, chondrocytes, myoblasts, corneal and vascular endothelial cells from either bovine or human origin, vascular smooth muscle cells and lens epithelial cells. Basic FGF was also shown to substitute for platelet-derived growth factor in its ability to support the proliferation of fibroblasts exposed to plasma-supplemented medium. Consistent with its ability to stimulate the proliferation of bovine and vascular endothelial cells, basic FGF has a similar activity in vivo on capillary endothelial cells; therefore, basic FGF is also considered an angiogenic factor.
It is this basic form of pituitary and brain FGF with which the applicants' invention is concerned.
Because basic FGF has important value both for promoting cell growth in vitro and as a therapeutic agent, it would be desirable to be able to obtain sizable amounts of substantially pure basic FGF. This is particularly the case where the substance is to be administered to a human, as administration of unseparated impurities in extracts is to be avoided whenever possible.
A need exists for improved, simplified methods of purifying basic FGF.